Microsomal triglyceride transfer protein (MTP) is essential for apoB-lipoprotein assembly. MTP is typically studied using a multi-step radiolabel assay. Recently, we have developed a simple fluorescence assay for MTP. However, this assay needs special equipment and expertise in lipid biochemistry.
Our aim i s to simplify the assay and commercialize it in the form of a kit consisting of only two components: a master mix and purified MTP. The master mix will contain all the ingredients necessary for MTP activity. Our hypothesis is that the commercial success of the MTP kit would depend on the stability of the assay ingredients and simplicity of the procedure. To achieve this goal, we propose the following specific aims: (1) Prepare or purify donor and acceptor vesicles in the presence of NaCI, (2) Add BSA to these vesicles during their purification, (3) Increase the shelf life of vesicles by inhibiting lipid oxidation, (4) Explore the possibility of mixing and storing donor and acceptor vesicles together, (5) Formulate a master mix that contains all the ingredients necessary for MTP assay. The outcome of all these manipulations will be evaluated by studying the incorporation of fluorescent lipids into donor vesicles, blanks, totals, % of lipid transferred, and specific activity of MTP. There are several advantages to a stable and user-friendly MTP assay kit. A kit with longer half-life is expected to be active after its arrival at the user's desk and will provide sufficient time for the investigators to perform their experiments. In addition, this may allow mass production of the kit resulting in cheaper prices rather than preparing the vesicles every time the company receives a purchase order. The major benefit of a simple, sensitive and reliable MTP assay is to the laboratories that do not have expertise in lipid biochemistry and to the companies who would like to automate their procedures to identify compounds that modulate MTP activity. In Phase II, we will simplify MTP purification, increase its stability, and develop a commercialization plan for the MTP assay kit.
